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Background: Caffeine is regularly utilized for its advantages, which incorporate expanded watchfulness. It has reactions, nonetheless, for example, palpitations and withdrawal indications that incorporate migraines and languor. Tertiary instruction frequently expects understudies to read for broadened hours, particularly during times of expanded remaining task at hand preceding tests and assessments. Medicinal understudies, who need to ace an extremely enormous volume of scholastic work in a constrained timeframe, are no special case. This examination was led to decide the pattern of caffeine utilization among medicinal understudies and furthermore the reactions related with it. Methodology: It is a cross-sectional study. The present examination was directed among 3rd year M.B.B.S understudies at Sree Balaji Medical College and Hospital, Chennai. Around 100 restorative understudies took an interest in the investigation. A concise early on introduction was given to understudies about the method of reasoning and organization of the investigation. The information was gathered independently from anyone else evaluation poll accessible in English. Results: The examination demonstrated that lion's share of restorative understudies where low customers of caffeine during standard days though during test days, the measure of utilization moves more toward moderate admission class. The most common reason and situation behind caffeine admission was to battle laziness and keep them progressively aware and of concentrate for the test. The manifestation they confronted frequently was loss of rest and during test days, different central nervous system related impacts were likewise noted. Conclusion: The frequency of caffeine use among medical students increases during exam days while it remains moderate to low on regular days and the most common side effect associated with such intake was found to be nervousness and loss of sleep. [ABSTRACT FROM AUTHOR]

Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

SARS-CoV-2 continues to pose a threat to public health. Current therapeutics remain limited to direct acting antivirals that lack distinct mechanisms of action and are already showing signs of viral resistance. The virus encodes an ADP-ribosylhydrolase macrodomain (Mac1) that plays an important role in the coronaviral lifecycle by suppressing host innate immune responses. Genetic inactivation of Mac1 abrogates viral replication in vivo by potentiating host innate immune responses. However, it is unknown whether this can be achieved by pharmacologic inhibition and can therefore be exploited therapeutically. Here we report a potent and selective lead small molecule, AVI-4206, that is effective in an in vivo model of SARS-CoV-2 infection. Cellular models indicate that AVI-4206 has high target engagement and can weakly inhibit viral replication in a gamma interferon- and Mac1 catalytic activity-dependent manner; a stronger antiviral effect for AVI-4206 is observed in human airway organoids. In an animal model of severe SARS-CoV-2 infection, AVI-4206 reduces viral replication, potentiates innate immune responses, and leads to a survival benefit. Our results provide pharmacological proof of concept that Mac1 is a valid therapeutic target via a novel immune-restoring mechanism that could potentially synergize with existing therapies targeting distinct, essential aspects of the coronaviral life cycle. This approach could be more widely used to target other viral macrodomains to develop antiviral therapeutics beyond COVID-19., Competing Interests: A.R.R, P.J., R.L.G., T.T., M.R., J.S.F., G.J.C., B.K.S., R.J.N, A.A., M.D., P.C.O., Y.D.P., N.K., M.O., T.Y.T., R.S., F.Z.B., and M.M. are listed as inventors on a patent application describing small molecule macrodomain inhibitors, which includes compounds described herein. T.Y.T and M.O. are listed as inventors on a patent application filed by the Gladstone Institutes that covers the use of pGLUE to generate SARS-CoV-2 infectious clones and replicons. The Krogan laboratory has received research support from Vir Biotechnology, F. Hoffmann-La Roche and Rezo Therapeutics. N.J.K. has financially compensated consulting agreements with Maze Therapeutics and Interline Therapeutics. He is on the Board of Directors and is President of Rezo Therapeutics and is a shareholder in Tenaya Therapeutics, Maze Therapeutics, Rezo Therapeutics, GEn1E Lifesciences and Interline Therapeutics. B.K.S is co-founder of BlueDolphin LLC, Epiodyne Inc, and Deep Apple Therapeutics, Inc., and serves on the SRB of Genentech, the SAB of Schrodinger LLC, and the SAB of Vilya Therapeutics. M.O. is a cofounder of Directbio and board member of InVisishield. A.R.R. is a co-founder of TheRas, Elgia Therapeutics, and Tatara Therapeutics, and receives sponsored research support from Merck, Sharp and Dohme. A.A. is a co-founder of Tango Therapeutics, Azkarra Therapeutics and Kytarro; a member of the board of Cytomx, Ovibio Corporation, Cambridge Science Corporation; a member of the scientific advisory board of Genentech, GLAdiator, Circle, Bluestar/Clearnote Health, Earli, Ambagon, Phoenix Molecular Designs, Yingli/280Bio, Trial Library, ORIC and HAP10; a consultant for ProLynx, Next RNA and Novartis; receives research support from SPARC; and holds patents on the use of PARP inhibitors held jointly with AstraZeneca from which he has benefited financially (and may do so in the future). J.S.F. is a consultant to, shareholder of, and receives sponsored research support from Relay Therapeutics.

Objective: Workers' compensation guidelines may reinforce unhelpful thoughts regarding symptoms that are known to increase symptom intensity and magnitude of incapability., Methods: One guideline commonly used (the Official Disability Guidelines) was reviewed regarding carpal tunnel syndrome. For 15 statements, we created an alternative statement based on a set of consensus principles for health, value, and quality in care. One hundred eight upper extremity surgeons of the Science of Variation Group reviewed both versions of the statements to indicate their preference., Results: Surgeons preferred seven revised statements and five guideline statements and were neutral on three statements. Favored revisions related to more accurate discernment of symptoms that are clearly related to idiopathic median neuropathy and representative of severity of pathology., Conclusions: There may be important mental health considerations for care under a work claim, such as unhelpful thoughts or distress regarding symptoms, which are not adequately considered by the Official Disability Guidelines., Competing Interests: Disclosures: S.G., S.R., and T.J.C. certify that they have no commercial associations (eg, consultancies, stock ownership, equity interest, patent/licensing arrangements, etc) that might pose a conflict of interest in connection with the submitted article. One of the authors (J.M.M.) is an unpaid, voluntary reviewer for Official Disability Guidelines and the American College of Occupational and Environmental Medicine. One of the authors (D.R.) certifies that he, or a member of his immediate family, has or may receive payment or benefits from Skeletal Dynamics (less than US $100,000), Wright Medical for elbow implants (less than US $10,000), deputy editor for Clinical Orthopaedics and Related Research, Universities and Hospitals, and lawyers outside of the submitted work., (Copyright © 2022 American College of Occupational and Environmental Medicine.)

Bacterial cells and their associated plasmids and bacteriophages encode numerous small proteins of unknown function. One example, the 73-amino-acid protein TraR, is encoded by the transfer operon of the conjugative F plasmid of Escherichia coli. TraR is a distant homolog of DksA, a protein found in almost all proteobacterial species that is required for ppGpp to regulate transcription during the stringent response. TraR and DksA increase or decrease transcription initiation depending on the kinetic features of the promoter by binding directly to RNA polymerase without binding to DNA. Unlike DksA, whose full activity requires ppGpp as a cofactor, TraR is fully active by itself and unaffected by ppGpp. TraR belongs to a family of divergent proteins encoded by proteobacterial bacteriophages and other mobile elements. Here, we experimentally addressed whether other members of the TraR family function like the F element-encoded TraR. Purified TraR and all 5 homologs that were examined bound to RNA polymerase, functioned at lower concentrations than DksA, and complemented a dksA -null strain for growth on minimal medium. One of the homologs, λ Orf73, encoded by bacteriophage lambda, was examined in greater detail. λ Orf73 slowed host growth and increased phage burst size. Mutational analysis suggested that λ Orf73 and TraR have a similar mechanism for inhibiting rRNA and r-protein promoters. We suggest that TraR and its homologs regulate host transcription to divert cellular resources to phage propagation or conjugation without induction of ppGpp and a stringent response. IMPORTANCE TraR is a distant homolog of the transcription factor DksA and the founding member of a large family of small proteins encoded by proteobacterial phages and conjugative plasmids. Reprogramming transcription during the stringent response requires the interaction of DksA not only with RNA polymerase but also with the stress-induced regulatory nucleotide ppGpp. We show here that five phage TraR homologs by themselves, without ppGpp, regulate transcription of host promoters, mimicking the effects of DksA and ppGpp together. During a stringent response, ppGpp independently binds directly to, and inhibits the activities of, many proteins in addition to RNA polymerase, including translation factors, enzymes needed for ribonucleotide biosynthesis, and other metabolic enzymes. Here, we suggest a physiological role for TraR-like proteins: bacteriophages utilize TraR homologs to reprogram host transcription in the absence of ppGpp induction and thus without inhibiting host enzymes needed for phage development.

Transcription initiation requires formation of the open promoter complex (RPo). To generate RPo, RNA polymerase (RNAP) unwinds the DNA duplex to form the transcription bubble and loads the DNA into the RNAP active site. RPo formation is a multi-step process with transient intermediates of unknown structure. We use single-particle cryoelectron microscopy to visualize seven intermediates containing Escherichia coli RNAP with the transcription factor TraR en route to forming RPo. The structures span the RPo formation pathway from initial recognition of the duplex promoter in a closed complex to the final RPo. The structures and supporting biochemical data define RNAP and promoter DNA conformational changes that delineate steps on the pathway, including previously undetected transient promoter-RNAP interactions that contribute to populating the intermediates but do not occur in RPo. Our work provides a structural basis for understanding RPo formation and its regulation, a major checkpoint in gene expression throughout evolution., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

TraR and its homolog DksA are bacterial proteins that regulate transcription initiation by binding directly to RNA polymerase (RNAP) rather than to promoter DNA. Effects of TraR mimic the combined effects of DksA and its cofactor ppGpp, but the structural basis for regulation by these factors remains unclear. Here, we use cryo-electron microscopy to determine structures of Escherichia coli RNAP, with or without TraR, and of an RNAP-promoter complex. TraR binding induced RNAP conformational changes not seen in previous crystallographic analyses, and a quantitative analysis revealed TraR-induced changes in RNAP conformational heterogeneity. These changes involve mobile regions of RNAP affecting promoter DNA interactions, including the βlobe, the clamp, the bridge helix, and several lineage-specific insertions. Using mutational approaches, we show that these structural changes, as well as effects on σ 70 region 1.1, are critical for transcription activation or inhibition, depending on the kinetic features of regulated promoters., Competing Interests: JC, SG, CC, AC, EC, RG, WR, SD No competing interests declared, (© 2019, Chen et al.)

The stringent response to nutrient deprivation is a stress response found throughout the bacterial domain of life. Although first described in proteobacteria for matching ribosome synthesis to the cell's translation status and for preventing formation of defective ribosomal particles, the response is actually much broader, regulating many hundreds of genes-some positively, some negatively. Utilization of the signaling molecules ppGpp and pppGpp for this purpose is ubiquitous in bacterial evolution, although the mechanisms employed vary. In proteobacteria, the signaling molecules typically bind to two sites on RNA polymerase, one at the interface of the β' and ω subunits and one at the interface of the β' secondary channel and the transcription factor DksA. The β' secondary channel is targeted by other transcription regulators as well. Although studies on the transcriptional outputs of the stringent response date back at least 50 years, the mechanisms responsible are only now coming into focus.

σ S is an alternative sigma factor, encoded by the rpoS gene, that redirects cellular transcription to a large family of genes in response to stressful environmental signals. This so-called σ S general stress response is necessary for survival in many bacterial species and is controlled by a complex, multifactorial pathway that regulates σ S levels transcriptionally, translationally, and posttranslationally in Escherichia coli It was shown previously that the transcription factor DksA and its cofactor, ppGpp, are among the many factors governing σ S synthesis, thus playing an important role in activation of the σ S stress response. However, the mechanisms responsible for the effects of DksA and ppGpp have not been elucidated fully. We describe here how DksA and ppGpp directly activate the promoters for the anti-adaptor protein IraP and the small regulatory RNA DsrA, thereby indirectly influencing σ S levels. In addition, based on effects of DksA N88I , a previously identified DksA variant with increased affinity for RNA polymerase (RNAP), we show that DksA can increase σ S activity by another indirect mechanism. We propose that by reducing rRNA transcription, DksA and ppGpp increase the availability of core RNAP for binding to σ S and also increase transcription from other promoters, including P dsrA and P iraP By improving the translation and stabilization of σ S , as well as the ability of other promoters to compete for RNAP, DksA and ppGpp contribute to the switch in the transcription program needed for stress adaptation. IMPORTANCE Bacteria spend relatively little time in log phase outside the optimized environment found in a laboratory. They have evolved to make the most of alternating feast and famine conditions by seamlessly transitioning between rapid growth and stationary phase, a lower metabolic mode that is crucial for long-term survival. One of the key regulators of the switch in gene expression that characterizes stationary phase is the alternative sigma factor σ S Understanding the factors governing σ S activity is central to unraveling the complexities of growth, adaptation to stress, and pathogenesis. Here, we describe three mechanisms by which the RNA polymerase binding factor DksA and the second messenger ppGpp regulate σ S levels., (Copyright © 2017 American Society for Microbiology.)

The Escherichia coli F element-encoded protein TraR is a distant homolog of the chromosome-encoded transcription factor DksA. Here we address the mechanism by which TraR acts as a global regulator, inhibiting some promoters and activating others. We show that TraR regulates transcription directly in vitro by binding to the secondary channel of RNA polymerase (RNAP) using interactions similar, but not identical, to those of DksA. Even though it binds to RNAP with only slightly higher affinity than DksA and is only half the size of DksA, TraR by itself inhibits transcription as strongly as DksA and ppGpp combined and much more than DksA alone. Furthermore, unlike DksA, TraR activates transcription even in the absence of ppGpp. TraR lacks the residues that interact with ppGpp in DksA, and TraR binding to RNAP uses the residues in the β' rim helices that contribute to the ppGpp binding site in the DksA-ppGpp-RNAP complex. Thus, unlike DksA, TraR does not bind ppGpp. We propose a model in which TraR mimics the effects of DksA and ppGpp together by binding directly to the region of the RNAP secondary channel that otherwise binds ppGpp, and its N-terminal region, like the coiled-coil tip of DksA, engages the active-site region of the enzyme and affects transcription allosterically. These data provide insights into the function not only of TraR but also of an evolutionarily widespread and diverse family of TraR-like proteins encoded by bacteria, as well as bacteriophages and other extrachromosomal elements., Competing Interests: The authors declare no conflict of interest.

The alternative sigma factor σ E is a key component of the Escherichia coli response to cell envelope stress and is required for viability even in the absence of stress. The activity of σ E increases during entry into stationary phase, suggesting an important role for σ E when nutrients are limiting. Elevated σ E activity has been proposed to activate a pathway leading to the lysis of nonculturable cells that accumulate during early stationary phase. To better understand σ E -directed cell lysis and the role of σ E in stationary phase, we investigated the effects of elevated σ E activity in cultures grown for 10 days. We demonstrate that high σ E activity is lethal for all cells in stationary phase, not only those that are nonculturable. Spontaneous mutants with reduced σ E activity, due primarily to point mutations in the region of σ E that binds the -35 promoter motif, arise and take over cultures within 5 to 6 days after entry into stationary phase. High σ E activity leads to large reductions in the levels of outer membrane porins and increased membrane permeability, indicating membrane defects. These defects can be counteracted and stationary-phase lethality delayed significantly by stabilizing membranes with Mg 2+ and buffering the growth medium or by deleting the σ E -dependent small RNAs (sRNAs) MicA, RybB, and MicL, which inhibit the expression of porins and Lpp. Expression of these sRNAs also reverses the loss of viability following depletion of σ E activity. Our results demonstrate that appropriate regulation of σ E activity, ensuring that it is neither too high nor too low, is critical for envelope integrity and cell viability. IMPORTANCE The Gram-negative cell envelope and cytoplasm differ significantly, and separate responses have evolved to combat stress in each compartment. An array of cell envelope stress responses exist, each of which is focused on different parts of the envelope. The σ E response is conserved in many enterobacteria and is tuned to monitor pathways for the maturation and delivery of outer membrane porins, lipoproteins, and lipopolysaccharide to the outer membrane. The activity of σ E is tightly regulated to match the production of σ E regulon members to the needs of the cell. In E. coli , loss of σ E results in lethality. Here we demonstrate that excessive σ E activity is also lethal and results in decreased membrane integrity, the very phenotype the system is designed to prevent., (Copyright © 2017 American Society for Microbiology.)

Choline is an essential nutrient for humans which plays an important role in structural integrity and signaling functions. Docosahexaenoic acid (DHA) is a polyunsaturated fatty acid, highly enriched in cell membranes of the brain. Dietary intake of choline or DHA alone by pregnant mothers directly affects fetal brain development and function. But no studies show the efficacy of combined supplementation of choline and DHA on fetal neurodevelopment. The aim of the present study was to analyze fetal neurodevelopment on combined supplementation of pregnant dams with choline and DHA. Pregnant dams were divided into five groups: normal control [NC], saline control [SC], choline [C], DHA, and C + DHA. Saline, choline, and DHA were given as supplements to appropriate groups of dams. NC dams were undisturbed during entire gestation. On postnatal day (PND) 40, brains were processed for Cresyl staining. Pups from choline or DHA supplemented group showed significant ( p < 0.05) increase in number of neurons in hippocampus when compared to the same in NC and SC groups. Moreover, pups from C + DHA supplemented group showed significantly higher number of neurons ( p < 0.001) in hippocampus when compared to the same in NC and SC groups. Thus combined supplementation of choline and DHA during normal pregnancy enhances fetal hippocampal neurodevelopment better than supplementation of choline or DHA alone., Competing Interests: There is no conflict of interests among all coauthors regarding doing this study or publishing it.

The development of integrated instrumentation for universal bioassay systems serves as a key goal for the lab-on-a-chip community. The programmable bio-nano-chip (p-BNC) system is a versatile multiplexed and multiclass chemical- and bio-sensing system for bioscience and clinical measurements. The system is comprised of two main components, a disposable cartridge and a portable analyzer. The customizable single-use plastic cartridges, which now can be manufactured in high volumes using injection molding, are designed for analytical performance, ease of use, reproducibility, and low cost. These labcard devices implement high surface area nano-structured biomarker capture elements that enable high performance signaling and are index-matched to real-world biological specimens. This detection modality, along with the convenience of on-chip fluid storage in blisters and self-contained waste, represents a standard process to digitize biological signatures at the point-of-care. A companion portable analyzer prototype has been developed to integrate fluid motivation, optical detection, and automated data analysis, and it serves as the human interface for complete assay automation. In this report, we provide a systems-level perspective of the p-BNC universal biosensing platform with an emphasis on flow control, device integration, and automation. To demonstrate the flexibility of the p-BNC, we distinguish diseased and non-case patients across three significant disease applications: prostate cancer, ovarian cancer, and acute myocardial infarction. Progress towards developing a rapid 7 minute myoglobin assay is presented using the fully automated p-BNC system.

Horizontal gene transfer by conjugation plays a major role in bacterial evolution, allowing the acquisition of new traits, such as virulence and resistance to antibacterial agents. With the increased antibiotic resistance in bacterial pathogens, a better understanding of how bacteria modulate conjugation under changing environments and the genetic factors involved is needed. Despite the evolutionary advantages conjugation may confer, the process can be quite stressful for the donor cell. Here, we characterize the ability of TraR, encoded on the episomal F' plasmid, to upregulate the σ(E) extracytoplasmic stress pathway in Escherichia coli. TraR, a DksA homolog, modulates transcription initiation through the secondary channel of RNA polymerase. We show here that TraR activates transcription directly; however, unlike DksA, it does so without using ppGpp as a cofactor. TraR expression can stimulate the σ(E) extracytoplasmic stress response independently of the DegS/RseA signal transduction cascade. In the absence of TraR, bacteria carrying conjugative plasmids become more susceptible to external stress. We propose that TraR increases the concentrations of periplasmic chaperones and proteases by directly activating the transcription of σ(E)-dependent promoters; this increased protein folding capacity may prepare the bacterium to endure the periplasmic stress of sex pilus biosynthesis during mating., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The E. coli alternative sigma factor, σ(E) , transcribes genes required to maintain the cell envelope and is activated by conditions that destabilize the envelope. σ(E) is also activated during entry into stationary phase in the absence of envelope stress by the alarmone (p)ppGpp. (p)ppGpp controls a large regulatory network, reducing expression of σ(70) -dependent genes required for rapid growth and activating σ(70) -dependent and alternative sigma factor-dependent genes required for stress survival. The DksA protein often potentiates the effects of (p)ppGpp. Here we examine regulation of σ(E) by (p)ppGpp and DksA following starvation for nutrients. We find that (p)ppGpp is required for increased σ(E) activity under all conditions tested, but the requirement for DksA varies. DksA is required during amino acid starvation, but is dispensable during phosphate starvation. In contrast, regulation of σ(S) is (p)ppGpp- and DksA-dependent under all conditions tested, while negative regulation of σ(70) is DksA- but not (p)ppGpp-dependent during phosphate starvation, yet requires both factors during amino acid starvation. These findings suggest that the mechanism of transcriptional regulation by (p)ppGpp and/or DksA cannot yet be explained by a unifying model and is specific to individual promoters, individual holoenzymes, and specific starvation conditions., (© 2014 John Wiley & Sons Ltd.)

The widespread use of antimicrobials has increased the occurrence of multidrug resistant microbes. The commonest mechanism of antimicrobial resistance in Enterobacteriaceae is production of β-lactamases such as metallo-β-lactamases (MBL) and extended spectrum β-lactamases (ESBL). Few studies have used a molecular approach to characterize the prevalence of β-lactamases. Here, the prevalence of different β-lactamases was characterized by performing three multiplex PCRs targeting genes similar to those described in earlier publications. Antimicrobial susceptibility tests for all isolates were performed using the agar dilution method. β-lactamase was detected in 72% of the isolates, the detection rate being 64% in 2011 and 75% in 2012. The isolates were highly resistant to carbapenems such as meropenem and imipenem and susceptible to colistin and tigecycline. In this study, 22% of isolates contained both MBL and ESBL. ESBL was detected more frequently in Escherichia coli isolates, whereas carbapenemase was detected more frequently in Klebsiella pneumoniae isolates. These findings suggest the spread of multi-resistant ESBL and MBL producers in the community. Our results have implications for patient treatment and also indicate the need for increased surveillance and molecular characterization of isolates., (© 2013 The Societies and Wiley Publishing Asia Pty Ltd.)

Activated sludge from the Stickney Water Reclamation Plant of the Metropolitan Water Reclamation District of Greater Chicago was adapted in the laboratory to either benzoate or 2-chlorobenzoate as the sole carbon source in sequencing batch reactors with a 48-h feed-aerate-settle-draw cycle and a mean cell residence time (MCRT) of 10 days. Benzoate degradation increased by more than 80-fold after 7 MCRTs compared to unadapted activated sludge. A greater than 15-fold increase in 2-chlorobenzoate metabolism occurred after adaptation for about 5-7 MCRTs. For each substrate the maximum rate measured for adapted cultures was near or above the highest previously reported in the literature. For both adapted and unadapted sludges, benzoate metabolism was considerably faster than that of 2-chlorobenzoate, and for both substrates the rate of metabolism increased incrementally with time of adaptation. As expected, addition of the benzoate-adapted sludge to unadapted sludge enhanced the latter's ability to degrade benzoate., (Copyright © 2009 Elsevier GmbH. All rights reserved.)